Inhibition of in vivo [(3)H]MK-801 binding by NMDA receptor open channel blockers and GluN2B antagonists in rats and mice.

N-methyl-D-aspartate (NMDA) receptor antagonists, including open channel blockers and GluN2B receptor subtype selective antagonists, have been developed for the treatment of depression. The current study investigated effects of systemically administered NMDA channel blockers and GluN2B receptor antagonists on NMDA receptor activity in rodents using in vivo [(3)H]MK-801 binding. The receptor occupancy of GluN2B antagonists was measured using ex vivo [(3)H]Ro 25-6981 binding. Ketamine, a NMDA receptor channel blocker, produced a dose/exposure- and time-dependent inhibition of in vivo [(3)H]MK-801 binding that was maximal at ~100%. The complete inhibition of in vivo [(3)H]MK-801 binding was also observed with NMDA receptor channel blockers, AZD6765 (Lanicemine) and MK-801 (Dizocilpine). CP-101,606 (Traxoprodil), a GluN2B antagonist, produced a dose/exposure- and time-dependent inhibition of in vivo [(3)H]MK-801 binding that was maximal at ~60%. Partial inhibition was also observed with other GluN2B antagonists including MK-0657 (CERC-301), EVT-101, Ro 25-6981 and radiprodil. For all GluN2B antagonists tested, partial [(3)H]MK-801 binding inhibition was achieved at doses saturating GluN2B receptor occupancy. Combined treatment with ketamine (10mg/kg, i.p.) and Ro 25-6981(10mg/kg, i.p.) produced a level of inhibition of in vivo [(3)H]MK-801 binding that was similar to treatment with either agent alone. In conclusion, this in vivo [(3)H]MK-801 binding study shows that NMDA receptor activity in the rodent forebrain can be inhibited completely by channel blockers, but only partially (~60%) by GluN2B receptor antagonists. At doses effective in preclinical models of depression, ketamine may preferentially inhibit the same population of NMDA receptors as Ro 25-6981, namely those containing the GluN2B subunit.

Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice.

Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n=5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 µM) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner.

Effects of combined, multiple stressors on pyridostigmine-induced acute toxicity in rats.

A number of studies have evaluated the possibility that stress-induced changes in blood-brain barrier permeability enhanced the central effects of the carbamate acetylcholinesterase inhibitor, pyridostigmine. We previously found relatively little evidence of stress-induced changes in the acute toxicity of pyridostigmine in rats using a variety of restraint, forced running and forced swimming stress conditions. In this study, we evaluated the effects of sequential pre-exposure to multiple stressors on the acute toxicity of pyridostigmine. Rats (n = 8 per treatment group) were either un-stressed or stressed by restraint (60 min), forced running (60 min, 15 m/min, 6 degrees incline) and forced swimming (15 min), and then given either vehicle (saline, 1 ml/kg, po) or pyridostigmine (30 mg/kg, po) immediately after the final stressor. Functional signs of cholinergic toxicity (involuntary movements, autonomic dysfunction) were recorded at 0.5, 1 and 2 h after dosing. Body temperature was measured both before stress and 2 h after dosing. Rats were sacrificed immediately after 2-h functional observations to collect tissues (whole blood, diaphragm, frontal cortex, hippocampus and cerebellum) for measurement of cholinesterase activity. Stressed rats treated with pyridostigmine exhibited higher lethality (2/8) compared to unstressed rats given pyridostigmine (0/8). Pyridostigmine elicited classical signs of cholinergic toxicity, but the rats that died did not show increased cholinergic signs and no significant differences in cholinergic signs were noted between treatment groups. Cholinesterase activity was significantly inhibited in blood (47-50%) and diaphragm (80%) following pyridostigmine exposure regardless of stress conditions. Slight but significant inhibition (11-15%) of cerebellar cholinesterase activity was observed following pyridostigmine exposure, but inhibition was not influenced by stress. We conclude that while acute lethality from pyridostigmine may be increased by combined, multiple stressors, increased lethality does not appear due to enhanced cholinergic toxicity or via increased cholinesterase inhibition in either central or peripheral tissues.